Within the plasma membrane, the cytofacial and lumenal/exofacial leaflets of the bilayer are known to be composed of different phospholipid species. This unequal distribution of phospholipids within the bilayer is referred to as membrane phospholipid asymmetry. This asymmetry is coordinates a number of cellular functions ranging from membrane curvature, to secretory function, and intra- and intercellular signaling. Disruption of phospholipid asymmetry has been linked to neurological dysfunction, blood disorders, cholestasis, and type 2 diabetes. However, the means through which a cell maintains phospholipid asymmetry of the plasma membrane and internal organelles is poorly understood. One critical regulator of membrane phospholipid asymmetry is a family of enzymes called phospholipid flippases, or P4-type ATPases. My research plan will use forward genetic strategies and directed enzyme evolution to: i) determine the P4- ATPase residues used to discriminate phospholipid backbone, ii) define the molecular basis for phosphatidylethanolamine headgroup recognition, and iii) test the primary structural mechanism responsible for substrate protection and passage through the membrane. Elucidating these mechanisms will be critical to understanding how the cell sets, maintains, disrupts, and repairs phospholipid asymmetry of its membranes. These molecular findings will be critical for the design of new enzyme technologies for the perturbation and examination membrane asymmetry effectors within the nervous system, lymphocytes, vasculature, and hepatic system. Finally, we anticipate that a molecular understanding of substrate recognition and coordination will facilitate the production of pharmaceutical therapies for disease treatment.